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Epidemiological and microbiological study of an outbreak of infectious keratoconjunctivitis in sheep
T. Naglić1, D. Hajsig2, J. Frey3, B.Šeol1, K. Busch2, M. Lojkić4
1
Department of Microbiology and Infectious Diseases, Veterinary Faculty University of Zagreb, 10000 Zagreb,
Heinzelova 55, Croatia
2PLIVA
- Zagreb, Ulica grada Vukovara 49, 10000 Zagreb, Croatia
3
Institute for Veterinary Bacteriology, Länggasstrasse 122, CH-3012 Berne, Switzerland
4
Croatian Veterinary Institute, Savska 143, 10000 Zagreb, Croatia
Summary
Following the import of several thousand sheep from Australia and New Zealand to Croatia during 1995, a great
many native sheep that had been in contact with imported animals acquired a severe ocular disease closely
resembling ovine infectious keratoconjunctivitis (OIKC). In affected flocks glucose-fermenting mycoplasma
were isolated from 48% and Branhamella ovis from 58% of conjunctival swabs. As representatives, 12 of 42
culturally and biochemically identical isolates were identified as Mycoplasma conjunctivae by PCR. From the
conjunctivas of two animals M. conjunctivae and M. arginini were isolated in mixed culture. For many reasons
most farmers removed imported animals from their flocks and only sporadic cases of OIKC were recognized in
1996. At the end of 1997 six flocks clinically free from OIKC but affected with the disease in the course of 1995
and five flocks with no history of the severe ocular disease underwent clinical and microbiological examinations
and all were found free of M. conjunctivae infection. At the time, Branhamella ovis was cultured almost
exclusively from sheep originating from flocks affected with OIKC during 1995 and/or 1996. Customarily it was
found in pure culture or as the predominant bacterial species, and frequently was accompanied by mild
conjunctivitis. Its implication in the pathogenesis of OIKC or conjunctivitis, respectively, requires further
investigation. There were no microbiologically confirmed new cases of OIKC during 1998 and 1999. Because of
the costs of medication and need for special nursing, anxiety of the farmers caused by severity of clinical signs,
and other inconveniences, there is a strong case for regulations to prevent the import of OIKC in the countries
free of infection.
Key words: Mycoplasma conjunctivae, Mycoplasma arginini, Branhamella ovis, ovine infectious
keratoconjunctivitis
Introduction
Ovine infectious keratoconjunctivitis (OIKC), also described as "pink eye" (Hopkirk 1934), is an acute
contagious disease occurring throughout the world (Egwu 1991, Jones 1991). It is characterized by inflammation
of the conjunctiva and cornea, often resulting in a period of temporal or even persistent blindness (Egwu and
others 1989, Greig 1989). Although lambs as young as 5-10 days can become ill (Jones and others 1976), adult
animals experience more severe symptoms (Greig 1989). In many cases ocular changes regress spontaneously,
but relapses are not unusual. An outbreak of OIKC can be expected at any season of the year, particularly during
the pasture season (Nicolet and Freundt 1975). Because of frequent relapses, regardless of treatment, and the
need for special nursing during the period of reduced vision or total blindness, the disease is of equal concern to
veterinarians and farmers (Egwu and others 1989, Egwu 1991). Although OIKC is clinically well defined and
easily recognized, its aetiology remains uncertain (Jones and others 1976, Mayer and others 1996). Numerous
microorganisms have been suggested as aetiologic agents, but the cause and possible predisposing factors of the
disease are still disputed (Egwu 1991). Among a great variety of microorganisms that have been incriminated as
causative agents special attention has been focused on Branhamella (B.) ovis, Chlamydia (C.) psittaci and
Mycoplasma (M.) conjunctivae. Recent investigations indicate that M. conjunctivae could be the primary
aetiologic agent of OIKC (Egwu 1991, Giacometti and others 1998). This paper reports microbiological and
epidemiological observations during the first occurrence of OIKC in Croatia.
Case history
In early 1995, approximately 6,000 sheep (Corriedale, Comeback, Crossbreed I., Poll Dorset, Border Leicester)
from Australia and New Zealand were donated to Croatia. After transportation, sheep were kept on an isolated
farm for 30 days and during quarantine animals were serologically tested for brucellosis, listeriosis, Q-fever and
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FMD. Although there was a history of an mild ocular disease during the prolonged transportation OIKC was not
suspected. After quarantine sheep were distributed to farmers all over the country. Native and imported animals
were kept either together or in separate flocks, about 20 to 40 animals in each. During spring and summer of the
same year a great many native sheep that had been in contact with the imported animals developed signs of a
severe ocular disease closely resembling OIKC.
At the time of the first monitoring, 20% to 80% of indigenous animals had ocular changes involving one or both
eyes. The signs of the disease varied from mild conjunctivitis with hyperaemia of palpebral conjunctiva and clear
ocular discharge observed in some cases to marked keratoconjunctivitis in most of them. Severely diseased
animals showed signs of blepharospasm and marked photophobia. They frequently stood with their eyes closed
or bumped into barriers when moving. In more advanced cases mucopurulent keratitis with opacity and blood
vessels migrating into cornea and corneal ulceration were seen. At the time of our observation, a most of
imported animals appeared healthy, or showed signs of mild conjunctivitis. Keratoconjunctivitis with copious
ocular discharge and marked conjunctival hyperaemia in imported sheep occurred only sporadically. The
reported outbreaks of the disease in native sheep were strongly associated with their mixing with the imported
animals. Those unexposed directly to the imported animals remained healthy even if housed in adjacent pens. In
some cases the animals were probably infected when grazing common pasture.
At the beginning of the epizootic only topical or systemic treatment of affected sheep was predominantly applied
using tetracycline hydrochloride ophthalmic ointment (PLIVA Zagreb) and oxytetracycline dihydrate
(Geomycin® Retard, PLIVA Zagreb), respectively. The topical treatment was predominantly effective only in
mildly affected animals and if tetracycline ointment was used two or three times daily during one week. Five or
six days after the initial improvement, clinical signs usually recurred requiring further treatment. Similar effects
were achieved with single or triple parenteral administration of only long acting oxytetracycline. The most
effective was a combination of daily topical application of oxytetracycline eye ointment and triple (on days 0, 3
and 6) intramuscular injection of oxytetracycline dihydrate (226,5 mg/10 kg bodyweight per day). Treated
animals usually recovered in three to six days and relapsed more rarely.
For many reasons, in the course of 1996 most of the farmers slaughtered survived imported sheep and only
sporadic outbreaks of OIKC occurred among indigenous sheep during this year. At the time of second
monitoring at the end of 1997, all 11 flocks inspected, located in different regions of Croatia, were clinically free
from OIKC. No new cases were diagnosed officially during 1998 and 1999.
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Materials and methods
First monitoring (1995). From May to December 1995 conjunctival swabs were taken for
microbiological examination from 101 native and imported sheep, including two lambs and two rams, of
different ages and clinical status. The animals originated from 14 flocks located in different parts of
Croatia; 88 were from 12 OIKC affected flocks and 13 were from 2 healthy flocks which had had no
contact with infected animals. Most of the affected flocks had been treated with antibiotics before
sampling.
Second monitoring (1997). During the last three months of 1997, six currently healthy flocks which had
been affected with OIKC in the course of 1995, and five flocks with no history of the severe ocular
disease underwent clinical and microbiological examinations. The flocks, with 20 to 116 animals in
each, were located in central, northern and western parts of Croatia. The sheep in the flocks were of
different native breeds (Pramenka, Cigaja-Suffolk, Solcava Sheep, Istrian Sheep, Wuertemberg crossbreed). Imported animals were completely removed from all but one previously affected flock, in which
eleven imported animals were kept together with 105 native sheep. In each of the previously affected
flocks examined, farmers could identify at least some animals which showed typical ocular changes
during 1995 or 1996. The animals from the flocks with no history of OIKC had never been in contact
with imported animals. At the time of monitoring all sheep inspected were clinically free of OIKC but
about 10% in each flock showed the signs of mild conjunctivitis. The highest number (about 30%) of
animals with conjunctivitis was found in a flock with native and imported sheep kept together. At that
time, conjunctival swabs were taken for microbiological examination from 67 animals that originated
from 6 previously infected flocks, and from 38 animals derived from the 5 flocks with no history of
OIKC. The age of animals varied from several months to five years.
Methods. Sheep blood agar plates and PPLO agar and broth (Hayflick 1965) were inoculated with
ocular swabs immediately after sampling. Cultivation and identification of isolated bacteria and
mycoplasmas were performed according to established diagnostic techniques (Razin and Tully 1983,
Holt and others 1994). In the identification of bacteria, special attention was paid to branhamella,
Escherichia coli, staphylococci, streptococci and pseudomonas. Other opportunistic and saprophytic
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microflora were identified only provisionally if more than 5 colonies were grown up. At the first and
second monitoring, the smears prepared from ocular swabs from 20 sheep were stained by Giemsa
and modified Ziehl-Neelsen methods for microscopic detection of chlamydia (Stephenson and others
1974, Greig 1989). Additionally, at the time of first monitoring, the ocular materials from 12 diseased
sheep were inoculated into 7-day old chicken embryos and yolk sac impression smears were stained
by the above methods. Molecular identification of Mycoplasma conjunctivae was made by a PCR
method which amplifies the specific parts of the rrs gene of M. conjunctivae (Giacommetti and others
1999).
Results
At the first monitoring, glucose-fermenting mycoplasmas were cultivated from 42 (48%) of 88
conjunctival swabs from affected flocks. Two of these isolates were contaminated with argininehydrolyzing strains. Where ocular swabbing had not preceded the therapy, the rate of infected animals
in individual flocks was 77%. Although rarely clear clinical changes, a higher percentage of imported
(60%) than of native (38%) sheep was infected with mycoplasmas.
(Table 1)
In flocks with OIKC, mycoplasmas were isolated from clinically affected and unaffected animals but
were never found in the eyes of the animals originating from native flocks with no history of OIKC.
Mycoplasmas isolated from 42 animals grew in brownish colonies. They were glucose-positive and
arginine-negative, digitonin-sensitive, film and spots- and phosphatase-negative. Two strains
separated from mixed cultures were also digitonin-sensitive, phosphatase-negative, but hydrolyzed
arginine and did not ferment glucose, and their colonies were not brownish. Twelve of glucosefermenting mycoplasma isolates were further analyzed by PCR amplification which included
amplification of the entire rrs gene with universal prokaryotic primers, amplification of parts of rrs gene
specific to M. conjunctivae and a PCR method for the identification of M. agalactiae (Gonzales and
others 1995, Giacometti and others 1999). These 12 isolates were shown to be M. conjunctivae by the
amplification of the specific 748 bp PCR fragment (Fig. 1). Therefore, it can be assumed that most of
the other isolates with the same cultural and physiological characteristics also belonged to this
species. Two arginine-positive isolates were identified as M. arginini.
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B. ovis was recovered from 51 (58%) of 88 conjunctival swabs examined from affected flocks. It
occurred at a higher percentage in imported (85%) than in native sheep (35%). Thirteen (33%) of 39
clinically affected native sheep and 4 (33%) of 12 apparently healthy animals were infected with B.
ovis. The bacterium was found also in 8 (89%) of 9 clinically affected imported sheep and in 20 (61%)
of 33 apparently healthy animals. It was not found in any of the 13 native sheep from two flocks free of
OIKC. All isolates were non-glucose fermenting Gram-negative cocci, growing in "S" form, catalaseand oxidase-positive, urease-negative and -haemolytic (sheep erythrocytes). Other bacteria
belonging to genera Staphylococcus, Streptococcus, Bacillus or Escherichia were found only
occasionally in affected or unaffected ovine eyes, predominantly in mixed cultures. Chlamydiae were
absent in all cases.
At the second monitoring at the end of 1997, colonies suspected to be Mycoplasma sp. grew up from
conjunctival swabs of four native sheep that originated from a flock affected with OIKC during 1995.
Two isolates were slow-growing and two were fast-growing, glucose-positive and arginine-negative
strains. The isolates did not require serum for growth and grew at room temperature. Slow-growing
strains were isolated from eyes with signs of mild conjunctivitis in pure culture or together with B. ovis.
One fast-growing strain was isolated in pure culture from an animal with mild conjunctivitis, and the
other with coliforms from apparently healthy eyes. The isolates were not serologically identified, but
from the in cultural and biochemical characteristics they obviously did not belong to M. conjunctivae.
The overall results of bacteriological examinations are presented in Table 2.
(Table 2)
In the flocks affected with OIKC during 1995, B. ovis was customarily found in pure culture or as the
predominant bacterial species, frequently accompanied by mild conjunctivits. Branhamellas were
isolated more often from animals less than one year old (44%) than from those aged 1-3 years (33%)
and over 3 years (22%). The conjunctivitis was characterized by serous lachrymation and mild
hyperaemia of palpebral and bulbar vessels. On microscopical evidence, chlamydiae were not
detected in any specimen examined.
Discussion
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The microbiological and epidemiological investigations and clinical observations during the first
epizootic of OIKC in Croatia support the thesis that OIKC is primarily a mycoplasmal disease caused
by M. conjunctivae as described previously (Nicolet and Freundt 1975, Egwu and others 1989). This
microorganism was found in diseased as well as in apparently healthy animals, but always in
association with occurrence of OIKC in the flock. Mycoplasmal infections were commonly
accompanied by B. ovis isolated almost exclusively from sheep affected with OIKC or which originated
from affected flocks. Its implication in pathogenesis of OIKC as an associated causative agent or as a
secondary pathogen requires further investigation. Clinical and microbiological observations indicate
that B. ovis might be the only microbial agent sporadically causing mild conjunctivitis.
All investigations clearly indicated that the epizootic of OIKC in native sheep was related to the import
of infected animals. Introduction of imported sheep and rams into the flocks and direct contact with
them were mainly responsible for the appearance of the disease in native sheep.
The first
observations raised expectations that prompt removal of imported animals and acquired resistance of
native sheep would result in eradication of OIKC from Croatia. Our negative results at the second
monitoring, however, cannot exclude the possibility of new cases of OIKC with waning flock immunity.
Therefore, methods more sensitive than isolation of mycoplasmas by cultivation will be needed to
exclude animals carrying M. conjunctivae and to prevent reinfection of flocks with the agent
(Giacometti and others 1999). Taking account of the costs of medication and need for special nursing
during the period of reduced vision or total blindness, anxiety of the farmers caused by severity of
clinical signs, and other inconveniences, it is suggested that in countries free of infection small
ruminants should be subject to strict veterinary regulations in to prevent outbreaks of OIKC.
Acknowledgments
This study is part of the European COST Action 826 on "Ruminants mycoplasmoses" and was
supported by the Croatian Ministry of Science and Technology
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References
Egwu, G. O., Faull, W. B., Bradbury, J. M. & Clarkson, M. J. (1989) Ovine infectious
keratoconjunctivitis: a microbiological study of clinically unaffected and affected sheep's eye with
special reference to Mycoplasma conjunctivae. Veterinary Record 125, 253-256.
Egwu, G. O. (1991) Ovine infectious keratoconjunctivitis: an update. Veterinary Bulletin 61, 547-559.
Giacometti, M., Nicolet, J., Frey, J., Krawinkler, M., Meier, W., Welle, M., Johansson, K-E. & Degiorgis,
M. P. (1998) Susceptibility of alpine ibex to conjunctivitis caused by inoculation of a sheep-strain of
Mycoplasma conjunctivae. Veterinary Microbiology 61, 279-288.
Giacometti, M., Nicolet, J., Johansson, K-E., Naglić, T., Degiorgis, M-P. & Frey, J. (1999) Detection
and identification of Mycoplasma conjunctivae in infectious keratoconjunctivitis by PCR based on the
16S rRNA gene. Journal of Veterinary Medicine B 46, 173-180.
Gonzales, Y. R. C., Bascunana, C. R., Bolske, G., Mattsson, J. G., Molina, C. F. & Johansson, K-E.
(1995) In vitro amplification of the 16s rRNA genes from Mycoplasma bovis and Mycoplasma
agalactiae by PCR. Veterinary Microbiology 47, 183-190.
Greig, A. (1989) Ovine keratokonjunctivitis. In Practice 11, 110-113.
Hayflick, L. (1965) Tissue cultures and mycoplasmas. Texas Reports on Biology and Medicine
Supplement 1. 23, 285-303.
Holt, J. G., Krieg, N. R., Sneath, P. H. A., Staley, J. T. & Williams, S. T. (1994) Bergey's Manual of
Determinative Bacteriology. 9th ed. Baltimore, Philadelphia, Hong Kong, London, Munich, Sydney,
Tokyo, Williams & Wilkins.
Hopkirk, C. S. M. (1934) Pink eye in sheep. New Zealand Journal of Agriculture 48, 224-225.
Jones, G. E., Foggie, A., Sutherland, A. & Harker, D. B. (1976) Mycoplasmas and ovine
keratoconjunctivitis. Veterinary Record 99, 137-141.
Jones, G. E. (1991) Infectious keratoconjunctivitis. In Diseases of sheep. Eds W. B. Martin I. D. Aitken.
London, Blackwell Scientific Publications, pp. 280-283.
Mayer, D., Nicolet, J., Giacometti, M., Schmitt, M., Wahli, T. & Meier, W. (1996) Isolation of
Mycoplasma conjunctivae from conjunctival swabs of alpine ibex (Capra ibex ibex) affected with
infectious keratoconjunctivitis. Journal of Veterinary Medicine B. 43, 155-161.
Nicolet, J. and Freundt, E. A. (1975) Isolations of Mycoplasma conjunctivae from chamois and sheep
affected with keratoconjunctivitis. Zentralblatt für Veterinar-Medizin B. 22, 302-307.
Razin, S. & Tully, J. G. (1983) Methods in Mycoplasmology. Vol.1. Mycoplasma characterization. New
York, London, Paris, San Diego, San Francisco, Sao Paulo, Sydney, Tokyo, Toronto, Academic Press.
Inc.
Stephenson, E. H., Storz, J. & Hopkins, J. B.(1974) Properties and frequency of isolation of
chlamydiae from eyes of lambs with conjunctivitis and polyarthritis. American Journal of Veterinary
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Table 1. Occurrence of bacteria and mycoplasmas in eyes from infected sheep flocks
--------------------------------------------------------------------------------------------------------------Microorganism
Native sheep (48*)
Imported sheep (40*)
Total (88*)
___________________________________________________________________
Mycoplasma sp.
18 (38%)
24 (60%)
42 (48%)
Branhamella ovis
17 (35%)
34 (85%)
51 (58%)
Other bacteria
10 (21%)
19 (48%)
29 (33%)
_______________________________________________________________
* Number of animals examined
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Table 2. Bacteria and mycoplasmas in eyes at the second monitoring of sheep flocks
--------------------------------------------------------------------------------------------------------------Microorganism
Flocks with history of
OIKC
(67 animals examined)
Flocks without history
Total
of OIKC
(38 animals examined)
(105)
--------------------------------------------------------------------------------------------------------------Mycoplasma sp.
0
0
0
Acholeplasma sp.
4 (6%)
0
4 (4%)
28 (27%)
B. ovis
27 (40%)
1 (3%)
E. coli and coliforms
3 (4%)
5 (13%)
8 (8%)
Bacillus sp.
10 (15%)
24 (63%)
34 (32%)
Staphylococcus sp.
17 (25%)
21 (55%)
38 (36%)
1 (3%)
6 (6%)
S. aureus
5 (7%)
Streptococcus sp.
11 (16%)
5 (13%)
16 (15%)
Negative
22 (33%)
8 (21%)
30 (29%)
---------------------------------------------------------------------------------------------------------------
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Figure 1. Confirmation of M. conjunctivae by PCR. Colonies suspected to be M. conjunctivae were lysed and
submitted to specific PCR analysis as described (Giacometti and others 1999). The photograph shows the
analysis of the PCR products from 7 strains on a 1% agarose gel, stained with ethidium bromide and
photographed upon exposure to UV light. + indicates the positive control M. conjunctivae type strain HRC/581T ,
 is the negative control. Std is the molecular mass standard phage  digested by Hind III. The arrow indicates
the specific band of DNA of 748 bp.
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Department of Microbiology and Infectious Diseases
Veterinary Faculty, University of Zagreb
Heinzelova 55, 10000 Zagreb, P. O. Box 190 Croatia
Prof. Dr. T. Naglic
Dr. Pathmini Nathan
The Veterinary Record
7 Mansfield Street London WIM 0AT
Great Britain
Dear Dr. Pathmini,
Thank you very much for your letter of 22nd October 1999 and information that our paper has been
recommended for publication in Veterinary Record. We can let you know that we have accepted with gratitude
all suggestions for amendments from scrutineers, and we hope that our manuscript is acceptable for printing now.
With kind regards
Yours sincerely
T. Naglić
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Dear Professor Frey,
I am glad to inform your that our paper “Epidemiological and microbiological study of an outbreak of infectious
keratoconjunctivitis in sheep” was evaluated by scrutineers as “...a good quality paper.......”, and it has been
accepted for publication in Veterinary Record.
With many greetings,
Yours sincerely
T. Naglić
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Department of Microbiology and Infectious Diseases
Veterinary Faculty University of Zagreb
10000 Zagreb, Heinzelova 55
Croatia
Zagreb, 11th May 2000
Dear, Mrs Elliott,
Thank you very much for the proofs. We accepted all suggestions. Edited version of 1 st paragraph in Results
with added word “affected” now reads correctly. “Film and spots” a known microbiological term describing a
cultural property of mycoplasma, always used without further explanation.
Sincerely yours
Prof. Dr T. Naglić
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